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INTRODUCTION

Excessive scar formation after deep burns is inevitable. In the healing process, hypertrophic scars, keloids, and contracture can

occur, which in turn cause emotional, social, and functional problems for the rest of the victim’s life.

The pathophysiology of the formation of hypertrophic scars, keloids, and contracture is not yet completely understood; for

Effects of Topical Tamoxifen on Wound Healing of Burned Skin in RatsShaban Mehrvarz1, Ali Ebrahimi1, Hedayat Sahraei2, Mohammad Hasan Bagheri1, Sima Fazili3, Shahram Manoochehry1, Hamid Reza Rasouli1

1Trauma Research Center, Baqiyatallah University of Medical Sciences, Tehran; 2Department of Physiology and Biophysics, and Behavioral Sciences Research Center, Baqiyatallah University of Medical Sciences, Tehran; 3Department of Pathology, Baqiyatallah University of Medical Sciences, Tehran, Iran

Background This study aimed to assess the effects of the topical application of tamoxifen on wound healing of burned skin in Wistar rats by evaluating 3 healing characteristics: fibrotic tissue thickness (FTT), scar surface area (SSA), and angiogenesis in the healed scar tissue.Methods Eighteen male Wistar rats were used in this study. A third-degree burn wound was made on the shaved animals’ back, measuring 2×2×2 cm. In the first group, a 2% tamoxifen ointment was applied to the wound twice daily for 8 weeks. The second group received a placebo ointment during the same period. The third group did not receive any treatment and served as the control group.Results The median (interquartile range= [Q1, Q3]) FTT was 1.35 (1.15, 1.62) mm, 1.00 (0.95, 1.02) mm, and 1.25 (0.8, 1.5) mm in the control, tamoxifen, and placebo groups, respectively (P=0.069). However, the FTT in the tamoxifen group was less than in the placebo and control groups. The median angiogenesis was 3.5 (3.00, 6.25), 8.00 (6.75, 9.25), and 7.00 (5.50, 8.25) vessels per high-power field for the control, tamoxifen, and placebo groups, respectively (P=0.067). However, the median angiogenesis was higher in the tamoxifen group than in the control group. No significant difference was observed in the mean SSA between the tamoxifen group and the control group (P=0.990). Conclusions Local application of tamoxifen increased angiogenesis and decreased the FTT, with no change in the SSA in burned skin areas. These effects are expected to expedite the wound healing process, reducing contracture and preventing hypertrophic scar and keloid formation.

Keywords Tamoxifen / Wound healing / Burns / Keloid / Rats

Correspondence: Shahram ManoochehryTrauma Research Center, Baqiyatallah University of Medical Sciences, 1435917541, Mollasadra Street, Sheikh Bahaee Street, Tehran, IranTel: +98-2188053766Fax: +98-2188037561E-mail: shahram.manoochehry@yahoo.com

We thank Dr. Ali Zamani, the Quality Assurance Manager of the Pars Darou Pharmaceutical Company for preparing the drug, and Dr. Mohammad Hosien Kalantar Motamedi for his kind assistance in editing this paper.

No potential conflict of interest relevant to this article was reported.

Received: 13 Nov 2016 • Revised: 8 Aug 2017 • Accepted: 29 Aug 2017pISSN: 2234-6163 • eISSN: 2234-6171 • https://doi.org/10.5999/aps.2017.44.5.378 • Arch Plast Surg 2017;44:378-383

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this reason, both surgical and non-surgical treatment modalities have yielded disappointing results.

Considering the high costs of excessive scar treatment despite various therapeutic strategies, controversies still exist regarding the best treatment. Therefore, it has been suggested that pre-venting excessive scar formation may be more effective [1].

Prevention of abnormal scarring after skin burns is a challeng-ing problem, and several therapies exist for the reduction of hy-pertrophic scar and keloid formation. Tamoxifen is a nonsteroi-dal antagonist of estradiol used extensively in the prevention and treatment of breast cancer. This drug reducing the produc-tion of collagen by decreasing transforming growth factor (TGF-β2), and can prevent fibroblast formation in wound heal-ing. It is also implicated in the reduction of hypertrophic scar and keloid formation. Previous studies have shown the effects of tamoxifen on inhibiting fibroblasts in Dupuytren contracture affecting the palmar fascia [2], keloids [3] and retroperitoneal fibrosis [4]. In an in vitro study, it was shown that exposure to tamoxifen in human dermal cells could decrease cell growth fac-tors (basic fibroblast growth factor and vascular endothelial growth factor), and it was suggested that the clinical use of tamoxifen may decrease scar formation [5]. A randomized con-trolled trial of 300 patients with a history of hypertrophic scar-ring showed that oral tamoxifen could significantly reduce hy-pertrophic scars after surgical incisions [6].

Tamoxifen prevents myofibroblast differentiation and may have a therapeutic role in chronic inflammatory conditions or cancer [7]. Prevention of keloid and hypertrophic scar forma-tion is still a challenging field, and several different therapeutic modalities have been reviewed by Berman et al. [8].

This study aimed to assess the effects of the topical application of tamoxifen on wound healing of burned skin in Wistar rats by evaluating 3 healing characteristics: fibrotic tissue thickness (FTT), scar surface area (SSA), and angiogenesis in healed scar tissue.

METHODS

Eighteen male Wistar rats with a mean weight of 250 to 300 g were used in this study. The rats were obtained from our hospi-tal animal experimental laboratory, and the study was conduct-ed in the same laboratory. The rats were randomly divided into 3 groups, all with an equal number of animals. All animals were anesthetized via intraperitoneal injection of ketamine (60 mg/kg), and a 3- × 3-cm area of dorsal hair was shaved (Fig. 1). A third-degree burn wound was made with a 2- × 2- × 2-cm stain-less steel cube (Fig. 2) that was placed in 96°C water for 3 min-utes and then was placed on the shaved area for 15 seconds (Fig. 3). All animals were kept under standard laboratory conditions and were provided with identical food and water during the study period. After recovery, rats were individually housed in separate cages. The cages were cleaned daily and kept free from infectious agents. In 2 of the groups, a 2% tamoxifen ointment or a placebo ointment was applied to the wound twice daily for 8 weeks. Each of the animals was kept in a separate cage to pre-vent licking of the applied ointment by other mice, so we did not use any cover or dressing over the wounds after applying the ointment.

In the first group, a 2% tamoxifen ointment was applied to the wound twice daily for 8 weeks. The second group received a

Rats were anaesthetized and the dorsal hair was shaved in a 3×3 cm area.

The designed instrument was heated in boiling water for 3 minutes at 96°C, and was put on the shaved area for 15 sec-onds to generate a third-degree burn wound.

Fig. 1. An anaesthetized rat with a shaved back Fig. 2. Generating a third-degree burn wound

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placebo ointment during the same period. The third group did not receive any treatment and served as the control group. Tamoxifen and the placebo ointment were provided in similar tubes. Both tamoxifen and the placebo ointment were pur-chased from the Iran Hormone Pharmaceutical Company, Teh-ran, Iran. The 2% tamoxifen and placebo ointments were pre-pared in 2 differently colored containers without any labels. This drug made only for our experiment by the pharmaceutical company Pars Darou. The ingredients of the placebo ointment were a combination of glyceryl monostearate, liquid paraffin, petrolatum, propylene glycol, cetyl alcohol, and isopropyl my-ristate.

The ointment was the vehicle only, with no active ingredient. Throughout the entire period of this study, none of the investi-gators knew which ointment was tamoxifen, and each animal had a private code number, which was used as an identification

code for pathology.The protocol of the study was approved by the Animal Medical

Ethics Committee of Baqiyatallah University of Medical Scienc-es (Ref. letter no. s/340/3/5694). In order to collect specimens, the animals were euthanized at 8 weeks after the burn injury was made, under a high concentration of chloroform (Fig. 4).

Histopathological assessmentThe wounds were photographed on the first and last day of the study. The SSA was calculated in square millimeters. For patho-logic evaluation, excisional biopsies from the thickest area of the scar were performed, and the amount of fibrosis and angiogene-sis were assessed via a microscopic examination. Hematoxylin and eosin staining was used to evaluate angiogenesis (vessels per high-power field, HPF) (Fig. 5), and Masson trichrome staining

A third-degree burn wound was generated in all rats.

Fig. 3. Rats after burn wound generation Hematoxylin and eosin staining was used to evaluate angiogenesis (×400).

Fig. 5. Evaluation of angiogenesis

Fig. 4. Wounds on the last day of the experiment

Gross view of wounds on the last day of the experiment. (A) A rat from the control group. (B) A rat from the placebo group. (C) A rat from the tamoxifen group. The arrows point to the wound scar sites.

A B C

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was used to measure the FTT in millimeters (Fig. 6). All mea-surements were conducted by 2 independent blinded patholo-gists. All participants in the project, including the laboratory staff, wound assessors, and the data analyzer were blinded to the group allocation and treatments.

Data analysisData analysis was performed using SPSS ver. 24.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics such as median and the interquartile range (IQR; Q1, Q3) were used to present the results. The Kruskal-Wallis test was performed to analyze the differences among the 3 groups. P-values of < 0.05 were consid-ered to indicate statistical significance. Pairwise comparisons between the groups were performed using the Dunn method.

RESULTS

A summary of the data analysis results and comparisons across groups is presented in Table 1. At the end of the study, the me-dian (IQR = [Q1, Q3]) FTT was 1.35 (1.15, 1.62) mm, 1.00

(0.95, 1.02) mm, and 1.25 (0.8, 1.5) mm in the control, tamoxi-fen, and placebo groups, respectively.

No significant difference was observed in the FTT across the groups (P = 0.069). However, the FTT in the tamoxifen group was less than in the control and placebo groups. The median (IQR = [Q1, Q3]) angiogenesis was 3.5 (3.00, 6.25), 8.00 (6.75, 9.25) and 7.00 (5.50, 8.25) vessels/HPF for the control, tamox-ifen, and placebo groups, respectively. The median angiogenesis was higher in the tamoxifen group than in the control group. However, these differences between the groups was not statisti-cally significant (P = 0.067).

The SSA was 89.00 (69.00, 112.50) mm2, 77.50 (52.00, 100.00) mm2, and 49.00 (39.00, 60.00) mm2 for the control, tamoxifen, and placebo groups, respectively. However, no sig-nificant difference was observed between the tamoxifen group and the control group (P = 0.990). However, the SSA was sig-nificantly smaller in the placebo group than in the tamoxifen group (P = 0.032).

DISCUSSION

This study found that local application of tamoxifen ointment could reduce excessive scar formation and wound contraction in third-degree burned skin. This effect may have been caused by inhibiting fibrin accumulation, facilitating the process of healing, and maintaining and increasing the blood supply to the wound.

The present study showed that tamoxifen reduced the thick-ness of the fibrotic tissue; however, this was not statistically sig-nificant, which might have been due to the limited number of samples. The clinical importance of this issue is that the preven-tion of hypertrophic scar and keloid formation is the most chal-lenging problem in patients with severe burns.

In the present study, a significant decrease was observed in the final surface area of the scar in the placebo group compared with

Masson trichrome staining was used to measure fibrotic tissue thickness (×400).

Fig. 6. Measurements of fibrotic tissue thickness

Items Fibrotic tissue (thickness, mm) Angiogenesis (vessels/HPF) Scar surface area (mm2)

Tamoxifen 1.00 (0.95, 1.02) 8.00 (6.75, 9.25) 77.50 (52.00, 100.00)Control 1.35 (1.15, 1.62) 3.50 (3.00, 6.25) 89.00 (69.00, 112.50)Placebo 1.25 (0.80, 1.50) 7.00 (5.50, 8.25) 49.00 (39.00, 60.00)P-value 0.069 0.067 0.028Pairwise comparisons by the Dunn method A versus B --a) --a) 0.990 A versus C --a) --a) 0.160 B versus C --a) --a) 0.032

Data are presented as median (IQR= [Q1, Q3]).HPF, high-power field; A, tamoxifen; B, control; C, placebo.a)Multiple comparisons were not performed because the Kruskal-Wallis test results were not significantly different.

Table 1. Comparison of tamoxifen with the control and placebo

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the tamoxifen group. It appears that wound coverage by the ointment in that group prevented the wound dehydration and facilitated wound re-epithelialization, which consequently in-creased fibroblast activity and reduced the final scar area. Inhibi-tion of wound contraction is clinically important because it would not only ameliorate problems such as pain, deformity, and limitations in body movements, but also reduce the need for surgical correction [9].

There was no statistically significant reduction in the final sur-face area of the scars in the tamoxifen group compared with the control group. This was most likely due to the inhibitory effect of tamoxifen on the TGF-β activation pathway, which leads to the inhibition of myofibroblast induction; these events ulti-mately reduce α-smooth muscle actin and extracellular matrix proteins (such as collagen and fibronectin) as suppliers of wound contraction power. Thus, wound contraction is reduced. Hu et al. have also shown that tamoxifen reduces wound con-traction by inhibiting the collagen network [10,11].

Tamoxifen pills (Nolvadex) are used to prevent and treat es-trogen receptor-positive types of breast cancer. A liquefied form of tamoxifen (Soltamox) is also available. Tamoxifen ointment is used mainly for experimental proposes. Tamoxifen can cause side effects, including weight gain, sexual problems, thrombo-embolic events, stroke, and neurocognitive problems [12]. These systemic side effects are important, and the long-term use of oral tamoxifen should therefore be avoided.

The mechanisms of the effects of tamoxifen are complex and have not been clearly defined. It binds to estrogen receptors and blocks the proliferative action of estrogen in mammary cells. This action of tamoxifen is induced by the synthesis of the cyto-kine TGF-β, which acts as a negative autocrine regulatory mole-cule [13].

Leventhal et al. [14], in a meta-analysis, reviewed 36 articles published about the outcomes of various types of treatment modalities, and concluded that there was no optimal treatment modality for the management of keloid and hypertrophic scars.

Topical applications for the treatment or prevention of post-surgical skin injuries are noninvasive and more comfortable, and also cost less than other modalities [15].

Mikulec et al. [16] proposed that tamoxifen may improve wound healing in keloids by reducing the expression of TGF-β1.

Foo and Tristani-Firouzi [17] reviewed topical modalities for the treatment and prevention of post-surgical hypertrophic scars, argued that there is no universally accepted treatment for the prevention or elimination of hypertrophic scars, and then recommended “massage therapy” for the reduction of post-burn scarring.

Based on the results of this study, it appears that the use of

tamoxifen locally on the burned skin of rats caused an increase in angiogenesis, decreased the FTT, and caused no significant change in the final scar size in the burned areas. These factors can expedite the wound healing process by reducing contracture and preventing hypertrophic scar and keloid formation. More-over, the local use of tamoxifen locally does not carry the risk of systemic side effects of the drug.

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